05_Dr. Rajesh Sawant_Grouping cross matching issues and troubleshoots.pdf

Cross matching to study and clear the concepts..

1. Grouping, Cross Matching:
Issues and Troubleshoots
Dr RAJESH B SAWANT MD,PDCR
Consultant-Transfusion Medicine, Histocompatibility
and Immunogenetics
Kokilaben Dhirubhai Ambani Hospital
Mumbai, India

2. Discovery

•Experiments with blood transfusions have been
carried out for hundreds of years. Many patients
have died and it was not until 1901, when the
Austrian Karl Landsteiner discovered human blood
groups, that blood transfusions became safer.
•Awarded the Nobel Prize in 1930.
•4 main types (A, B, O, AB)
•ABO gene located on long arm of chromosome 9

3. Precursor substance

H substance

A antigen B antigen AB antigen No antigen
The red cells of O group lack A&B antigen because
O gene is amorphous, so group O cells contain
only H substance.
FORMATION OF AB & H

4. Secretor/Nonsecretor
•Se & se are called secretor genes.
Persons secreting A/B, H substance in
saliva other body fluids are called
secretors.

Secretor 80% Se Se / Se se
Nonsecretor 20% se se.

5. ABO Typing: Procedure
•Cell/Forward Group
–Test Washed Cells With:
–Monoclonal Anti-A
–Monoclonal Anti-B
–Inert control
•Agglutination/hemolysis
is a positive result
•Serum/Reverse Group
–Test plasma/serum with:
–Known A
1 cells
–Known B cells
–Known O cells
•Reactions may be
weaker than cell group
ALWAYS Compare results of cell and serum grouping and then lable the unit

6. ABO Antibodies
•Generally IgM class antibodies
•For Group A and Group B persons the
predominant antibody class is IgM
•For Group O people the dominant
antibody class is IgG (with some IgM)
•React best at room temperature (22-
24
o
C) or below in vitro.
•Activates complement to completion
at 37
o
C
• Can cause acute Hemolytic
Transfusion reactions
•RBC Immune form: Predominantly
IgG

7. A and B Subgroup
•Differentiation between the A blood subgroups
•Reagent anti-A is a mixture of two Abs
The two Abs can be functionally separated by adsorption
with A2 cells.

Anti-A1-lectin: is another source of anti-A1.
lectins are seed extracts that agglutinate human cells
with some degree of specificity.
The seeds of the plant Dolichos biflorus serve as the
source of the anti-A1 lectin this reagent agglutinate A1 or
A1B cells but does not agglutinate A2 or A2B cells.

8. A and B Subgroup
•Other A subgroups: RBC of the A
int, A
3, Ax, A
y or A
cl. are
only rarely seen in transfusion practice.

•Subgroup of B: infrequent than the weaker subgroup of
A, identified by anti-B and anti-A,B. Subgroups B3 , Bx ,
Bm and Bcl .

9. ABO Discrepancies
•ABO discrepancies occur when there is no match in
results between forward and reverse grouping.

•ABO discrepancies are usually technical in nature and
can be simply resolved by correctly reporting the testing
and carefully checking reagents with meticulous reading
and recording of results.

10. ABO Discrepancies
•There are some ABO discrepancies that can happen due
to technical errors and may lead to false positive or false
negative reactions.

•False positive reactions are due to;
–Un-calibrated centrifuges
–Contaminated reagents
–Dirty tubes or glassware

11. ABO Discrepancies
•False negative reactions can be due to many causes:


–Failure to add serum or reagents
–Use of incorrect reagents or samples
–Cell suspension is too heavy or too light
–Inadequate identification of samples or test tubes

12. ABO Discrepancies
•Group I discrepancies
These discrepancies are between forward and reverse
grouping due to weak reaction or missing antibodies.

These kind of discrepancies are the most common.

The reason for the missing antibody or weak reaction is
that the patient has depressed antibody production or
cannot produce the ABO antibodies.

13. ABO Discrepancies
• This type of discrepancy can be seen in new born
infants, elderly patients.

•Patients with lymphoma.

•Patients using immunosuppressive drugs.

•Patients with immunodeficiency disease, BM transplant.

14. ABO Discrepancies
•Resolving discrepancies

•Eliminate all technical errors
•Enhancing the reaction in reverse grouping
•Incubate the patient’s serum with reagent cells at room
temp. for 15 mins.

15. ABO Discrepancies
Group II discrepancies
These discrepancies are between forward and reverse
grouping due to weak reaction or missing antigens.
Can be caused by some subgroups of A or subgroups of
B or both.

Also it can be present in patients with leukaemia and
Hodgkin’s disease.

To resolve the problem wash the patient’s cells with saline.

16. ABO Discrepancies
Group III discrepancies

These discrepancies are between forward and reverse
grouping due to protein or plasma abnormalities.

These can be caused by elevated levels of globulin from
certain diseases such as multiple myloma, hodgkin’s
lymphoma. Some are caused by Rouleaux formation.

17. ABO Discrepancies
•Rouleaux or red cells result from a stacking of
erythrocytes that adhere in a coin-link fashion giving the
appearance of agglutination.

•To resolve this kind of problem, washing the patient’s
red cells with saline or adding a drop or two of saline to
the tube in case of rouleaux formation.

•If the agglutination is true red cell clumping will remain.

•Cord blood must be washed 6-8 times in forward
grouping ONLY.

18. ABO Discrepancies
Group IV discrepancies
•These kind of discrepancies are between forward and
reverse groping due to miscellaneous problems.

•Polyagglutination can occur due to exposure of hidden
erythrocyte Ag. (T antigen) in patients with bacterial or
viral infection.

•Bacterial contamination in vitro or vivo produces an
enzyme that alters and exposes the hidden Ag. on red cell
leading to T activation.

19. ABO Discrepancies
•Some examples of discrepancies:

Example 1
Forward grouping: anti-A =0, anti-B =0, anti-AB= 0
Reverse grouping: A1 cells= 0, B cells =0
Blood group: O
Possible discrepancy:
Missing Ab or group I discrepancy

20. ABO Discrepancies
Example 2

Forward grouping: anti-A 4+,anti-B 2+,anti-AB 4+
Reverse grouping: A1 cells 4+, B cells 4+
Blood group : A
Possible discrepancy: Rouleaux formation

21. Significance of ABO Group
•ABO mismatched transfusions:
–Rare
–May be life threatening
–Can be caused by technical or clerical error
–Intravascular haemolysis
–More severe in group O patients

22. The H system
•H system has two genes H & h,
•one antigen H as precursor molecule on
which A&B are built
•O group has no A&B antigen but has
abundant H antigen.
•Amount of H antigen detected on red
cells in diminished ordered is
O>A
2>B>A
2B>A
1>A
1B

23. Oh Phenotype
•First discovered in Bombay
•Red cells lacks H, A and B antigens
•Identified by agglutination reaction with O cells in
reverse grouping, confirmed by compatible reaction
with known Oh red cells
•Absence of reaction with Anti-H lectin (Ulex
europeaus)
•Oh person must be transfused only with Oh blood
Anti-
A
Anti-
B
Anti-AB A cells B cells O cells
- - - + + +

25. Rh Blood Group System

26. Clinical Significance
•Formation of antibody – from exposure
through transfusion or pregnancy
•30-85% of D- person who receives
exposure will develop anti-D

27. Rh antigens
•Mid-1940s – four additional antigens –C, E, c, e
•Rh system
•Rh related antigens are 49, qualitative &
quantitative variations
•Five (D,C,E,c,e) account for 99% clinical issues
•Rh antigens are fully expressed at birth with
detection as early as 8 wks of gestation
•Present on Red cells only

28. Expression of D
•D- persons either lack
RHD or have
nonfunctional RHD
gene.
•Most D- are
homozygous for RHce
•RHCE rare in D- or D+

D Positive
D Negative

29. Scenarios of Discrepant results

44. Troubleshooting the Incompatible
Crossmatch

45. Trouble Shooting the Incompatible
Crossmatch

•Immediate Spin:
•Non-Immune Causes-
•Fibrin
•Rouleaux

46. Rouleaux


•Caused ? an e?ess of protein in the patient’s
plasma
•If present, it should first be detected in the
reverse grouping of non-group O patients
•Should react with all units tested, including
the A1 and B cells
•Will disperse with saline replacement

48. Non-Specific Cold Agglutinin


•Should have first been detected in the reverse
grouping of non-group O patients
•Should react with all donor units tested,
including the A1 and B cells
•Often caused by performing a xm with cold
plasma

49. Cold Antibody with Specificity


•Reacts with some of the units, possibly with
varying strengths
•May not have been detected in the grouping
or in the antibody screen
•Could be an anti-P1, -Lua, -Lea, -Leb, -M

50. How to Investigate a Cold Reacting
Antibody in the Crossmatch

52. Passive ABO Antibodies


•Will occur in non-group O patients only
•Patient has recent transfusion history of either
IVIgG or ABO mismatched platelets or
mismatched solid organ transplant
•Possible positive DAT and eluate that reacts
against A1 and/B cells
•Give group O blood until antibody disappears

53. New Alloantibody


•Some of the units are reacting with varying
strengths
•Patient may have a positive DAT
•Will require an antibody investigation
•May cause a delay in providing blood if
antigen negative units need to be sourced

54. AHG XM with a Strongly Incompatible
Unit

55. Antibody to Low Frequency Antigen


•Perform a direct antiglobulin test on the unit
•Recheck the antigen typing on the unit
•Review the antibody investigation, paying
particular attention to the exclusions
•If the exclusions are valid, give antigen-
negative, crossmatch compatible blood

56. Auto control is positive
If this occurs, perform the following:
•Do DAT
•Get medications list
•Get diagnosis
•Get transfusion history
•If DAT is positive and patient recently transfused,
do elution technique to remove the antibody
from transfused donor cells and identify its
specificity

57. Caution!!
•Negative Antibody Screening Cells, but records
show an antibody was previously present.
•In this case the titer of antibody has dropped
below detectable levels.
Steps to follow for this problem:
•Perform crossmatch through the antiglobulin,
Coombs, test.
•Type donor units for antigen. The donor units
must be negative for the antigen that the
recipient formerly had an antibody to.

58. Summary


•There are immune and non-immune reasons for
incompatibility in a crossmatch
•Consider passive ABO antibodies when problem solving
the incompatible crossmatch in non-group O patients
•One strongly incompatible unit in a crossmatch is
probably due to an antibody to a low frequency
antigen
•Consult with the Medial Diretor/patient’s ph?siian
before issuing incompatible blood

59. Thank You